RESUMO
Stimulation of the dorsal half of the rat periaqueductal gray (DPAG) with 60-Hz pulses of increasing intensity, 30-µA pulses of increasing frequency, or increasing doses of an excitatory amino acid elicits sequential defensive responses of exophthalmia, immobility, trotting, galloping, and jumping. These responses may be controlled by voltage-gated calcium channel-specific firing patterns. Indeed, a previous study showed that microinjection of the DPAG with 15 nmol of verapamil, a putative blocker of L-type calcium channels, attenuated all defensive responses to electrical stimulation at the same site as the injection. Accordingly, here we investigated the effects of microinjection of lower doses (0.7 and 7 nmol) of both verapamil and mibefradil, a preferential blocker of T-type calcium channels, on DPAG-evoked defensive behaviors of the male rat. Behaviors were recorded either 24 h before or 10 min, 24 h, and 48 h after microinjection. Effects were analyzed by both threshold logistic analysis and repeated measures analysis of variance for treatment by session interactions. Data showed that the electrodes were all located within the dorsolateral PAG. Compared to the effects of saline, verapamil significantly attenuated exophthalmia, immobility, and trotting. Mibefradil significantly attenuated exophthalmia and marginally attenuated immobility while facilitating trotting. While galloping was not attenuated by either antagonist, jumping was unexpectedly attenuated by 0.7 nmol verapamil only. These results suggest that T-type calcium channels are involved in the low-threshold freezing responses of exophthalmia and immobility, whereas L-type calcium channels are involved in the trotting response that precedes the full-fledged escape responses of galloping and jumping.
Assuntos
Bloqueadores dos Canais de Cálcio , Canais de Cálcio Tipo L , Canais de Cálcio Tipo T , Estimulação Elétrica , Mibefradil , Substância Cinzenta Periaquedutal , Verapamil , Animais , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/fisiologia , Masculino , Canais de Cálcio Tipo T/fisiologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo L/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Mibefradil/farmacologia , Verapamil/farmacologia , Ratos , Ratos Wistar , Microinjeções , Relação Dose-Resposta a DrogaRESUMO
The mechanical properties of living cells, including their shape, rigidity, and internal dynamics play a crucial role in their physiology and pathology. Still, the relations between the physiological cell state and its rigidity and surface vibrations remain poorly understood. Here, we have employed AFM measurements on T cells and found a negative relation between cell surface stiffness and its vibrations. Blocking T-type Ca++-channels using Mibefradil reduced cortical actin tension in these cells and enhanced their membrane vibrations and dissipation of intracellular mechanical work to the cell surroundings. We also found increased vibrations of cell membranes in five different malignant cells lines derived from T cell leukemia, lung, prostate, bladder, and melanoma cancers, as compared to their corresponding benign cells. This was demonstrated by utilizing TIRF microscopy in single cells and dynamic laser speckles measurements in an in vitro model of multiple cells in a tissue. Our results show that cell membrane vibrations and dissipation of mechanical work are higher in malignant cells relative to benign cells. Accordingly, these properties may be used to detect and monitor cellular and tissue malignancies.
Assuntos
Neoplasias , Vibração , Humanos , Membrana Celular/metabolismo , Mibefradil , Actinas/metabolismo , Linhagem CelularRESUMO
The acrosome is a lysosome-related vesicular organelle located in the sperm head. The acrosomal reaction (AR) is an exocytic process mediated by Ca2+ and essential for mammalian fertilization. Recent findings support the importance of acrosomal alkalinization for the AR. Mibefradil (Mib) and NNC 55-0396 (NNC) are two amphipathic weak bases that block the sperm-specific Ca2+ channel (CatSper) and induce acrosomal pH (pHa ) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pHa elevation increase the intracellular Ca2+ concentration ([Ca2+ ]i ) and trigger the AR by unknown mechanisms of Ca2+ transport. Here, we investigated the pathways associated with the pHa increase-induced Ca2+ signals using mouse sperm as a model. To address these questions, we used single-cell Ca2+ imaging, the lysosomotropic agent Gly-Phe-ß-naphthylamide (GPN) and pharmacological tools. Our findings show that Mib and NNC increase pHa and release acrosomal Ca2+ without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca2+ release caused by pHa rise. Inhibition of two-pore channel 1 (TPC1) channels reduced the [Ca2+ ]i increase stimulated by acrosomal alkalinization. In addition, blockage of Ca2+ release-activated Ca2+ (CRAC) channels diminished Ca2+ uptake triggered by pHa alkalinization. Finally, our findings contribute to understanding how pHa controls acrosomal Ca2+ efflux and extracellular Ca2+ entry during AR in mouse sperm. KEY POINTS: The acrosomal vesicle is a lysosome-related organelle located in the sperm head. The acrosome reaction (AR) is a highly regulated exocytic process mediated by Ca2+ , which is essential for fertilization. However, the molecular identity of Ca2+ transporters involved in the AR and their mechanisms to regulate Ca2+ fluxes are not fully understood. In mammalian sperm, acrosomal alkalinization induces intracellular Ca2+ concentration ([Ca2+ ]i ) increase and triggers the AR by unknown molecular mechanisms of Ca2+ transport. In this study, we explored the molecular mechanisms underlying Ca2+ signals caused by acrosomal alkalinization using mouse sperm as a model. TPC1 and CRAC channels contribute to [Ca2+ ]i elevation during acrosomal alkalinization. Our findings expand our understanding of how the acrosomal pH participates in the physiological induction of the AR.
Assuntos
Cálcio , Sêmen , Masculino , Animais , Camundongos , Cálcio/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Acrossomo/metabolismo , Mibefradil/metabolismo , Mibefradil/farmacologia , Concentração de Íons de Hidrogênio , Mamíferos/metabolismoRESUMO
Prostate cancer is often treated by perturbing androgen receptor signalling. CACNA1D, encoding CaV1.3 ion channels is upregulated in prostate cancer. Here we show how hormone therapy affects CACNA1D expression and CaV1.3 function. Human prostate cells (LNCaP, VCaP, C4-2B, normal RWPE-1) and a tissue microarray were used. Cells were treated with anti-androgen drug, Enzalutamide (ENZ) or androgen-removal from media, mimicking androgen-deprivation therapy (ADT). Proliferation assays, qPCR, Western blot, immunofluorescence, Ca2+-imaging and patch-clamp electrophysiology were performed. Nifedipine, Bay K 8644 (CaV1.3 inhibitor, activator), mibefradil, Ni2+ (CaV3.2 inhibitors) and high K+ depolarising solution were employed. CACNA1D and CaV1.3 protein are overexpressed in prostate tumours and CACNA1D was overexpressed in androgen-sensitive prostate cancer cells. In LNCaP, ADT or ENZ increased CACNA1D time-dependently whereas total protein showed little change. Untreated LNCaP were unresponsive to depolarising high K+/Bay K (to activate CaV1.3); moreover, currents were rarely detected. ADT or ENZ-treated LNCaP exhibited nifedipine-sensitive Ca2+-transients; ADT-treated LNCaP exhibited mibefradil-sensitive or, occasionally, nifedipine-sensitive inward currents. CACNA1D knockdown reduced the subpopulation of treated-LNCaP with CaV1.3 activity. VCaP displayed nifedipine-sensitive high K+/Bay K transients (responding subpopulation was increased by ENZ), and Ni2+-sensitive currents. Hormone therapy enables depolarization/Bay K-evoked Ca2+-transients and detection of CaV1.3 and CaV3.2 currents. Physiological and genomic CACNA1D/CaV1.3 mechanisms are likely active during hormone therapy-their modulation may offer therapeutic advantage.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Androgênios , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Nifedipino/farmacologia , Mibefradil/farmacologia , Linhagem Celular Tumoral , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Canais de Cálcio Tipo L/genéticaRESUMO
Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. Mice lacking Notch3 show deficient renal autoregulation. The aim of the study was to investigate the mechanisms involved in the Notch3-mediated control of renal vascular response. To this end, renal resistance vessels (afferent arterioles) were isolated from Notch3-/- and wild-type littermates (WT) and stimulated with angiotensin II (ANG II). Contractions and intracellular Ca2+ concentrations were blunted in Notch3-/- vessels. ANG II responses in precapillary muscle arterioles were similar between the WT and Notch3-/- mice, suggesting a focal action of Notch3 in renal vasculature. Abolishing stored Ca2+ with thapsigargin reduced Ca2+ responses in the renal vessels of the two strains, signifying intact intracellular Ca2+ mobilization in Notch3-/-. EGTA (Ca2+ chelating agent), nifedipine (L-type channel-blocker), or mibefradil (T-type channel-blocker) strongly reduced contraction and Ca2+ responses in WT mice but had no effect in Notch3-/- mice, indicating defective Ca2+ entry. Notch3-/- vessels responded normally to KCl-induced depolarization, which activates L-type channels directly. Differential transcriptomic analysis showed a major down-regulation of Cacna1h gene expression, coding for the α1H subunit of the T-type Ca2+ channel, in Notch3-/- vessels. In conclusion, renal resistance vessels from Notch3-/- mice display altered vascular reactivity to ANG II due to deficient Ca2+-entry. Consequently, Notch3 is essential for proper excitation-contraction coupling and vascular-tone regulation in the kidney.
Assuntos
Rim , Nifedipino , Receptor Notch3 , Animais , Camundongos , Angiotensina II/farmacologia , Arteríolas/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Rim/metabolismo , Mibefradil/metabolismo , Nifedipino/farmacologia , Resistência Vascular , Receptor Notch3/genética , Deleção de Genes , Camundongos KnockoutRESUMO
Population aging has led to increased sick sinus syndrome (SSS) incidence; however, no effective and safe medical therapy has been reported thus far. Yixin-Fumai granules (YXFMs), a Chinese medicine granule designed for bradyarrhythmia treatment, can effectively increase SSS patients' heart rate. Senescence-induced sinoatrial node (SAN) degeneration is an important part of SSS pathogenesis, and older people often show high levels of oxidative stress; reactive oxygen species (ROS) accumulation in the SAN causes abnormal SAN pacing or conduction functions. The current study observed the protective effects of YXFMs on senescent SAN and explored the relationship between the NRF-2/HO-1 pathway, SHOX2, and T-type calcium channels. We selected naturally senescent C57BL/6 mice with bradycardia to simulate SSS; electrocardiography, Masson's trichrome staining, and DHE staining were used to assess SAN function and tissue damage. Immunofluorescence staining and Western blotting were used to assay related proteins. In vitro, we treated human-induced pluripotent stem cell-derived atrial myocytes (hiPSC-AMs) and mouse atrial myocyte-derived cell line HL-1 with D-galactose to simulate senescent SAN-pacemaker cells. CardioExcyte96 was used to evaluate the pulsatile function of the hiPSC-AMs, and the mechanism was verified by DCFH-DA, immunofluorescence staining, RT-qPCR, and Western blotting. The results demonstrated that YXFMs effectively inhibited senescence-induced SAN hypofunction, and this effect possibly originated from scavenging of ROS and promotion of NRF-2, SHOX2, and T-type calcium channel expression. In vitro experiment results indicated that ML385, si-SHOX2, LDN193189, and Mibefradil reversed YXFMs' effects. Moreover, we, for the first time, found that ROS accumulation may hinder SHOX2 expression; YXFMs can activate SHOX2 through the NRF-2/HO-1 pathway-mediated ROS scavenging and then regulate CACNA1G through the SHOX2/BMP4/GATA4/NKX2-5 axis, improve T-type calcium channel function, and ameliorate the SAN dysfunction. Finally, through network pharmacology and molecular docking, we screened for the most stable YXFMs compound that docks to NRF-2, laying the foundation for future studies.
Assuntos
Canais de Cálcio Tipo T , Heme Oxigenase-1/metabolismo , Proteínas de Homeodomínio , Fator 2 Relacionado a NF-E2/metabolismo , Aceleração , Idoso , Animais , Galactose , Frequência Cardíaca , Proteínas de Homeodomínio/metabolismo , Humanos , Medicina Tradicional Chinesa , Mibefradil , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The role of T-type calcium channels is well established in excitable cells, where they preside over action potential generation, automaticity, and firing. They also contribute to intracellular calcium signaling, cell cycle progression, and cell fate; and, in this sense, they emerge as key regulators also in non-excitable cells. In particular, their expression may be considered a prognostic factor in cancer. Almost all cancer cells express T-type calcium channels to the point that it has been considered a pharmacological target; but, as the drugs used to reduce their expression are not completely selective, several complications develop, especially within the heart. T-type calcium channels are also involved in a specific side effect of several anticancer agents, that act on microtubule transport, increase the expression of the channel, and, thus, the excitability of sensory neurons, and make the patient more sensitive to pain. This review puts into context the relevance of T-type calcium channels in cancer and in chemotherapy side effects, considering also the cardiotoxicity induced by new classes of antineoplastic molecules.
Assuntos
Canais de Cálcio Tipo T , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Humanos , Mibefradil/farmacologiaRESUMO
OBJECTIVE: To observe the effect of mibefradil on skeletal muscle mass, function and structure in obese mice. METHODS: Fifteen 6-week-old C57BL/6 mice were randomized equally into normal diet group (control group), high-fat diet (HFD) group and high-fat diet +mibefradil intervention group (HFD +Mibe group). The grip strength of the mice was measured using an electronic grip strength meter, and the muscle content of the hindlimb was analyzed by X-ray absorptiometry (DXA). Triglyceride (TG) and total cholesterol (TC) levels of the mice were measured with GPO-PAP method. The cross-sectional area of the muscle fibers was observed with HE staining. The changes in the level of autophagy in the muscles were detected by Western blotting and immunofluorescence assay, and the activation of the Akt/mTOR signaling pathway was detected with Western blotting. RESULTS: Compared with those in the control group, the mice in HFD group had a significantly greater body weight, lower relative grip strength, smaller average cross sectional area of the muscle fibers, and a lower hindlimb muscle ratio (P < 0.05). Immunofluorescence assay revealed a homogenous distribution of LC3 emitting light red fluorescence in the cytoplasm in the muscle cells in HFD group and HFD+Mibe group, while bright spots of red fluorescence were detected in HFD group. In HFD group, the muscular tissues of the mice showed an increased expression level of LC3 II protein with lowered expressions of p62 protein and phosphorylated AKT and mTOR (P < 0.05). Mibefradil treatment significantly reduced body weight of the mice, lowered the expression level of p62 protein, and increased forelimb grip strength, hindlimb muscle ratio, cross-sectional area of the muscle fibers, and the expression levels of LC3 II protein and phosphorylated AKT and mTOR (P < 0.05). CONCLUSION: Mibefradil treatment can moderate high-fat diet-induced weight gain and improve muscle mass and function in obese mice possibly by activating AKT/mTOR signal pathway to improve lipid metabolism and inhibit obesityinduced autophagy.
Assuntos
Dieta Hiperlipídica , Proteínas Proto-Oncogênicas c-akt , Animais , Peso Corporal , Mibefradil/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: T-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases. METHODS: In this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-ß1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177. RESULTS: We demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-ß1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death. CONCLUSION: This study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.
Assuntos
Canais de Cálcio Tipo T , Células Mesangiais , Animais , Humanos , Células Mesangiais/metabolismo , Mibefradil/metabolismo , Mibefradil/farmacologia , Camundongos , Fosforilação , Ratos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Medulloblastoma (MB) is the most common malignant paediatric brain tumour. In our previous studies, we developed a novel 3D assay for MB cells that was used to screen a panel of plasma membrane calcium channel modulators for their effect on the 3D growth of D341 MB cells. These studies identified T-type (CaV3) channel inhibitors, mibefradil and NNC-55-0396 (NNC) as selective inhibitors of MB cell growth. Mibefradil was originally approved for the treatment of hypertension and angina pectoris, and recently successfully completed a phase I trial for recurrent high-grade glioma. NNC is an analogue of mibefradil with multiple advantages compared to mibefradil that makes it attractive for potential future clinical trials. T-type channels have a unique low voltage-dependent activation/inactivation, and many studies suggest that they have a direct regulatory role in controlling Ca2+ signalling in non-excitable tissues, including cancers. In our previous study, we also identified overexpression of CaV3.2 gene in MB tissues compared to normal brain tissues. In this study, we aimed to characterise the effect of mibefradil and NNC on MB cells and elucidate their mechanism of action. This study demonstrates that the induction of toxicity in MB cells is selective to T-type but not to L-type Ca2+ channel inhibitors. Addition of CaV3 inhibitors to vincristine sensitised MB cells to this MB chemotherapeutic agent, suggesting an additive effect. Furthermore, CaV3 inhibitors induced cell death in MB cells via apoptosis. Supported by proteomics data and cellular assays, apoptotic cell death was associated with reduced mitochondrial membrane potential and reduced ATP levels, which suggests that both compounds alter the metabolism of MB cells. This study offers new insights into the action of mibefradil and NNC and will pave the way to test these molecules or their analogues in pre-clinical MB models alone and in combination with vincristine to assess their suitability as a potential MB therapy.
Assuntos
Canais de Cálcio Tipo T , Neoplasias Cerebelares , Meduloblastoma , Apoptose , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Criança , Humanos , Meduloblastoma/tratamento farmacológico , Mibefradil/farmacologia , Mibefradil/uso terapêutico , Recidiva Local de Neoplasia , Vincristina/farmacologiaRESUMO
INTRODUCTION: The mucociliary transport function of the airway epithelium is largely dependent on ciliary beating. The control signal of ciliary beating is thought to be intracellular Ca2+. We herein investigated the expression of T-type voltage-gated calcium channel (VGCC), a generator of intracellular Ca2+ oscillation, in the human nasal mucosa. METHODS: The inferior turbinate was collected from patients with chronic hypertrophic rhinitis. The expression of T-type VGCC α1 subunits was examined by immunohistochemistry, transmission immunoelectron microscopy, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR). Participation of T-type VGCC in the ciliary beat regulation was examined by pharmacological inhibition tests using specific blockers of T-type VGCC in ex vivo measurements of the ciliary beat frequency (CBF) and ATP release and in intracellular Ca2+ imaging of isolated ciliated cells. RESULTS: Immunohistochemical staining showed the expressions of T-type VGCC α1 subunits, Cav3.1 and Cav3.3, on the surface of the epithelial cells. At the ultrastructural level, immunoreactivity for Cav3.1 was localized on the surface of the cilia, and that for Cav3.3 was localized in the cilia and at the base of the cilia. The existence of Cav3.1 and Cav3.3 was confirmed at the protein level by Western blot and at the transcriptional level by real-time RT-PCR. Specific blockers of T-type VGCC, mibefradil and NNC 55-0396, significantly inhibited CBF. These blockers also inhibited a CBF increase induced by 8-bromo-cAMP/8-bromo-cGMP and significantly lowered the intracellular Ca2+ level of isolated ciliated cells in a time-dependent manner. On the other hand, the ATP release from the nasal mucosa was not changed by mibefradil or NNC 55-0396. CONCLUSION: These results indicate that T-type VGCC α1 subunits, Cav3.1 and Cav3.3, exist at the cilia of the nasal epithelial cells and participate in the regulation of ciliary beating and that these channels act downstream of cAMP/cGMP.
Assuntos
Canais de Cálcio Tipo T , Cílios , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Cílios/fisiologia , GMP Cíclico , Células Epiteliais/metabolismo , Humanos , Mibefradil/metabolismo , Mibefradil/farmacologia , Mucosa Nasal/metabolismoRESUMO
Atrial fibrillation (AF) is associated with atrial conduction disturbances caused by electrical and/or structural remodelling. In the present study, we hypothesized that connexin might interact with the calcium channel through forming a protein complex and, then, participates in the pathogenesis of AF. Western blot and whole-cell patch clamp showed that protein levels of Cav1.2 and connexin 43 (Cx43) and basal ICa,L were decreased in AF subjects compared to sinus rhythm (SR) controls. In cultured atrium-derived myocytes (HL-1 cells), knocking-down of Cx43 or incubation with 30 mmol/L glycyrrhetinic acid significantly inhibited protein levels of Cav1.2 and Cav3.1 and the current density of ICa,L and ICa,T . Incubation with nifedipine or mibefradil decreased the protein level of Cx43 in HL-1 cells. Moreover, Cx43 was colocalized with Cav1.2 and Cav3.1 in atrial myocytes. Therefore, Cx43 might regulate the ICa,L and ICa,T through colocalization with calcium channel subunits in atrial myocytes, representing a potential pathogenic mechanism in AF.
Assuntos
Remodelamento Atrial , Canais de Cálcio/fisiologia , Conexina 43/fisiologia , Átrios do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Fibrilação Atrial/metabolismo , Remodelamento Atrial/fisiologia , Western Blotting , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/fisiologia , Linhagem Celular , Células Cultivadas , Conexina 43/metabolismo , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/fisiopatologia , Humanos , Mibefradil/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Nifedipino/farmacologia , Técnicas de Patch-ClampRESUMO
OBJECTIVE@#To observe the effect of mibefradil on skeletal muscle mass, function and structure in obese mice.@*METHODS@#Fifteen 6-week-old C57BL/6 mice were randomized equally into normal diet group (control group), high-fat diet (HFD) group and high-fat diet +mibefradil intervention group (HFD +Mibe group). The grip strength of the mice was measured using an electronic grip strength meter, and the muscle content of the hindlimb was analyzed by X-ray absorptiometry (DXA). Triglyceride (TG) and total cholesterol (TC) levels of the mice were measured with GPO-PAP method. The cross-sectional area of the muscle fibers was observed with HE staining. The changes in the level of autophagy in the muscles were detected by Western blotting and immunofluorescence assay, and the activation of the Akt/mTOR signaling pathway was detected with Western blotting.@*RESULTS@#Compared with those in the control group, the mice in HFD group had a significantly greater body weight, lower relative grip strength, smaller average cross sectional area of the muscle fibers, and a lower hindlimb muscle ratio (P < 0.05). Immunofluorescence assay revealed a homogenous distribution of LC3 emitting light red fluorescence in the cytoplasm in the muscle cells in HFD group and HFD+Mibe group, while bright spots of red fluorescence were detected in HFD group. In HFD group, the muscular tissues of the mice showed an increased expression level of LC3 II protein with lowered expressions of p62 protein and phosphorylated AKT and mTOR (P < 0.05). Mibefradil treatment significantly reduced body weight of the mice, lowered the expression level of p62 protein, and increased forelimb grip strength, hindlimb muscle ratio, cross-sectional area of the muscle fibers, and the expression levels of LC3 II protein and phosphorylated AKT and mTOR (P < 0.05).@*CONCLUSION@#Mibefradil treatment can moderate high-fat diet-induced weight gain and improve muscle mass and function in obese mice possibly by activating AKT/mTOR signal pathway to improve lipid metabolism and inhibit obesityinduced autophagy.
Assuntos
Animais , Camundongos , Peso Corporal , Dieta Hiperlipídica , Mibefradil/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Obesos , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
It has been shown that systemic and local administration of ultra-low dose morphine induced a hyperalgesic response via mu-opioid receptors. However, its exact mechanism(s) has not fully been clarified. It is documented that mu-opioid receptors functionally couple to T-type voltage dependent Ca+2 channels. Here, we investigated the role of T-type calcium channels, amiloride and mibefradil, on the induction of low-dose morphine hyperalgesia in male Wistar rats. The data showed that morphine (0.01 µg i.t. and 1 µg/kg i.p.) could elicit hyperalgesia as assessed by the tail-flick test. Administration of amiloride (5 and 10 µg i.t.) and mibefradil (2.5 and 5 µg i.t.) completely blocked low-dose morphine-induced hyperalgesia in spinal dorsal horn. Amiloride at doses of 1 and 5 mg/kg (i.p.) and mibefradil (9 mg/kg ip) 10 min before morphine (1 µg/kg i.p.) inhibited morphine-induced hyperalgesia. Our results indicate a role for T-type calcium channels in low dose morphine-induced hyperalgesia in rats.
Assuntos
Analgésicos Opioides/efeitos adversos , Canais de Cálcio Tipo T/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Morfina/efeitos adversos , Amilorida/farmacologia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Masculino , Mibefradil/farmacologia , Morfina/administração & dosagem , Morfina/antagonistas & inibidores , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Opioides muRESUMO
The effects and underlying mechanisms of mibefradil on gluconeogenesis and glycogenesis were investigated using insulin-resistant HepG2 human hepatocellular carcinoma cells and a mouse model of type 2 diabetes mellitus (T2DM). HepG2 cells were divided into one of four groups: control, palmitate (PA)-induced insulin-resistance (0.25 mM), low-concentration mibefradil (0.025 µM), or high-concentration mibefradil (0.05 µM). Glycogen synthesis and glucose consumption were evaluated in these HepG2 cells, and quantitative polymerase chain reaction (qPCR) and western blotting techniques were used to detect expression of forkhead box O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6-phosphatase (G6Pase). Intracellular calcium concentrations were determined using Fluo-4 AM, and phosphorylation levels of calmodulin-dependent protein kinase II (CaMKII), protein kinase B (Akt) and FoxO1were detected by western blotting. Immunofluorescence was used for the localization and quantification of FoxO1.In vitro results were verified using a mouse model of T2DM. In HepG2 cells and mouse liver tissues, mibefradil decreased PA-induced cytoplasmic calcium levels and CaMKII phosphorylation, but increased the phosphorylation of Akt and FoxO1, thereby contributing to the cytoplasmic localization of FoxO1. Additionally, mibefradil alleviated PA-induced glucose output and insulin resistance through increased glucose consumption and glycogen synthesis, while decreasing the expression of key gluconeogenesis enzymes, including PEPCK and G6Pase. Mibefradil may help to control blood sugar levels by reducing glucose output and insulin resistance, and the mechanism of action may involve the Ca2+-CaMKII-dependent Akt/FoxO1 signaling pathway.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Proto-Oncogênicas c-akt , Diabetes Mellitus Tipo 2 , Células Hep G2 , Humanos , MibefradilRESUMO
Pharmaceutical features of phenylalkylamine derivatives (PAAs) binding to calcium channels have been studied extensively in the past decades. Only a few PAAs have the binding specificity on calcium channels, for example, NNC 55-0396. Here, we created the homology models of human Cav 3.2, Cav 3.3 and use them as a receptor on the rigid docking tests. The nonspecific calcium channel blocker mibefradil showed inconsistent docking preference across four domains; however, NNC 55-0396 had a unique binding pattern on domain II specifically. The subsequent molecular dynamics (MD) simulations identified that Cav 3.1, Cav 3.2, and Cav 3.3 share domain II when Ca2+ appearing in the neighbor region of selective filters (SFs). Moreover, free-energy perturbation analysis suggests single mutation of lysine at P-loop domain III, or threonine at the P-loop domain II largely reduced the total amount of hydration-free energy in the system. All these findings suggest that P-loop and segment six domain II in the T-type calcium channels (TCCs) are crucial for attracting the PAAs with specificity as the antagonist.
Assuntos
Benzimidazóis/química , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo T/química , Ciclopropanos/química , Mibefradil/química , Modelos Moleculares , Naftalenos/química , HumanosRESUMO
TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.
Assuntos
Ameloblastos/metabolismo , Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Ameloblastos/citologia , Ameloblastos/efeitos dos fármacos , Anilidas/farmacologia , Animais , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Incisivo/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Mibefradil/farmacologia , Camundongos , Modelos Biológicos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Ratos , Tiadiazóis/farmacologiaRESUMO
Recent studies have illuminated that blocking Ca2+ influx into effector cells is an attractive therapeutic strategy for lung injury. We hypothesize that T-type calcium channel may be a potential therapeutic target for acute lung injury (ALI). In this study, the pharmacological activity of mibefradil (a classical T-type calcium channel inhibitor) was assessed in a mouse model of lipopolysaccharide- (LPS-) induced ALI. In LPS challenged mice, mibefradil (20 and 40 mg/kg) dramatically decreased the total cell number, as well as the productions of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). Mibefradil also suppressed total protein concentration in BALF, attenuated Evans blue extravasation, MPO activity, and NF-κB activation in lung tissue. Furthermore, flunarizine, a widely prescripted antimigraine agent with potent inhibition on T-type channel, was also found to protect mice against lung injury. These data demonstrated that T-type calcium channel inhibitors may be beneficial for treating acute lung injury. The important role of T-type calcium channel in the acute lung injury is encouraged to be further investigated.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Flunarizina/farmacologia , Lipopolissacarídeos/metabolismo , Mibefradil/farmacologia , Lesão Pulmonar Aguda/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Citocinas/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Cardiac hypertrophy causes heart failure and is associated with hyperglycemia in patients with diabetes mellitus. Mibefradil, which acts as a T-type calcium channel blocker, exerts beneficial effects in patients with heart failure. In this study, we explored the effects and mechanism of mibefradil on high-glucose-induced cardiac hypertrophy in H9c2 cells. H9c2 cells were incubated in a high-glucose medium and then treated with different concentrations of mibefradil in the presence or absence of the Akt inhibitor MK2206 or mTOR inhibitor rapamycin. Cell size was evaluated through immunofluorescence, and mRNA expression of cardiac hypertrophy markers (atrial natriuretic peptide, brain natriuretic peptide, and ß-myosin heavy chain) was assessed by using quantitative real-time polymerase chain reaction. Changes in the expression of p-PI3K, p-Akt, and p-mTOR were evaluated using Western blotting, and autophagosome formation was detected using transmission electron microscopy. Our results indicate that mibefradil reduced the size of H9c2 cells, decreased mRNA expression of atrial natriuretic peptide, brain natriuretic peptide, and ß-myosin heavy chain, and decreased the level of autophagic flux. However, MK2206 and rapamycin induced autophagy and reversed the effects of mibefradil on high-glucose-induced H9c2 cells. In conclusion, mibefradil ameliorated high-glucose-induced cardiac hypertrophy by activating the PI3K/Akt/mTOR pathway and inhibiting excessive autophagy. Our study shows that mibefradil can be used therapeutically to ameliorate cardiac hypertrophy in patients with diabetes mellitus.
Assuntos
Autofagia/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Cardiomegalia/prevenção & controle , Glucose/toxicidade , Mibefradil/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/ultraestrutura , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Encefálico/metabolismo , Fosforilação , Ratos , Transdução de SinaisRESUMO
Modes of interactions of small ligands with CYP3A4 have been defined using the Template established in our previous studies (DMPK. 34: 113-125 2019 and 34 351-364 2019). Interactions of polyaromatic hydrocarbons such as benzo[a]pyrene, pyrene and dibenzo[a,j]acridine were refined with the idea of Right-side movement of ligands at Rings A and B of Template. Expected formation of metabolites from the placements faithfully matched with experimentally observed sites of their metabolisms and also with preferred orders of regio-isomeric metabolite abundances in recombinant CYP3A4 system. In comparison of CYP3A4-ligand data with the placements on simulations, a futile sitting of non-substituted and free rotatable phenyl structures was suggested as a cause of poor oxidations of the phenyl parts of CYP3A4 ligands. These data were in turn indicative of the role of the rotation-ceasing action for the function. Typical inhibitors, ketoconazole, nicardipine, mibefradil and GF-I-1 shared mutuality on their sittings, in which the inhibitor molecules hold a CYP3A4 residue from dual sides on Template. In addition, clotrimazole would be stuck between facial- and rear-side walls of CYP3A4 and interact with ferric iron through nitrogen atom of the imidazole part. These data offered structural bases of CYP3A4-inhibitory actions of ligands.